Fermentation process

ABSTRACT

A process for the preparation of a target fermentation product comprising cultivation of an aerobic microorganism to produce such product, wherein in the microorganism the effi-ciency of the respiratory chain is increased, transformed microorganisms and novel nuc-leotide sequences.

The present invention relates to a process for the preparation of atarget fermentation pro-duct, to transformed microorganisms and to novelnucleotide sequences.

More particularly, the present invention provides a process for thepreparation of a target fermentation product comprising cultivation ofan aerobic microorganism to produce such product, wherein in themicroorganism the efficiency of the respiratory chain is increased.

Under aerobic conditions, generation of cellular energy in the form ofadenosine triphos-phate (ATP) occurs primarily by respiration, in whichthe electron flow from the reduced form of an electron carrier like NADHor FMNH2 to oxygen is coupled to proton trans-location across thecytoplasmic membrane. Respiration involves a set of membrane-asso-ciatedcompounds referred to as “respiratory chain” which are capable of beingreversibly oxidized and reduced. Many organisms, including Escherichia,Bacillus, Cyanobacter, Streptomyces, and Corynebacterium, e.g. E. coli,B. subtilis, B. amyloliquefaciens, B. licheni-formis, B. ammoniagenes,and C. glutamicum, have the ability to switch between alternativesubchains of the respiratory chain. These subchains have a commoncomponent, i.e. ubi-quinone or menaquinone, but different terminaloxidases. The respiratory chain of, e.g., Bacillus subtilis branchesinto the cytochrome C oxidase subchain and the quinol oxidase subchain.The terminal oxidase of the cytochrome C oxidase subchain is cytochromeaa3 oxidase encoded by ctaCDEF. The terminal oxidase of the quinoloxidase subchain is either cytochrome aa3 oxidase or cytochrome bdoxidase, encoded by qoxABCD and cydAB, res-pectively. In C. glutanlicumat least two subchains of the respiratory chain are present. Onesubchain consists of the cytochrome bc1c complex encoded by qcrABC andthe cytochrome aa3 oxidase encoded by ctaCDE. The other subchainconsists of cytochrome bd oxidase en-coded by cydAB.

The degree of coupling between electron flow and proton translocationand, thus, energe-tic efficiency differs between the subchains, not onlybetween the subchains as a whole but also between particular componentsof a subchain. For example, the above cytochrome aa3 oxidase is moreefficient than the above cytochrome bd oxidase, i.e. more protons aretranslocated per electron when cytochrome aa3 oxidase is involved.Naturally, cells utilise that subchain of the respiratory chain that isbest suited for growth under given environ-mental conditions.

To increase the efficiency of the respiratory chain a more energyefficient component of the respiratory chain may be introduced or,particularly in microorganisms having a natural capability to utilizealternative subchains of the respiratory chain, expression of a lessener-gy efficient component of a subchain may be prevented or reduced,thus directing the elec-tron flow to the more efficient subchain.

Examples for a microorganism having a natural capability to utilizealternative subchains of the respiratory chain which may be used in theprocess of the invention include mem-bers of the genera mentioned above.

As used herein, the term “a component of the respiratory chain isintroduced” refers to the introduction of a suitable polynucleotidesequence encoding said component into a micro-organism and therecombination of the polynucleotide sequence with the genomic DNA of themicroorganism by a single or double cross-over mechanism. Thepolynucleotide may contain transcriptional and translational signals,e.g. a promoter sequence and a ribosomal binding site, which are linkedto the gene encoding said component so as to allow expres-sion of thecomponent. For convenient selection of transformants of themicroorganism containing the polynucleotide sequence genes encoding anantibiotic resistance marker or a gene complementing a possibleauxotrophy of the microorganism may be encoded by the polynucleotidesequence as well. Furthermore, the polynucleotide may contain at the 5′and/or 3′ end DNA sequences of, e.g. at least 50 base pairs in lengthfrom the locus at which the polynucleotide sequence is to be introducedinto the genome of the micro-organism. The polynucleotide sequence maybe introduced at any locus within the genome of the microorganismprovided that no vital function for growth of the microorganism andproduction of the fermentation product is affected.

As used herein the term “expression of a component is prevented orreduced” refers to an alteration in the genome of a microorganism, whichinterferes with the biosynthesis of such component or leads to theexpression of a protein with an altered amino acid sequence whosefunction compared with the wild type counterpart with a non-alteredamino acid sequence is completely or partially destroyed. Theinterference may occur at the transcriptional, translational orpost-translational level. The alteration in the genome of themicroorganism may be obtained e.g. by replacing through a single ordouble cross-over recombination a wild type DNA sequence by a DNAsequence containing the alteration. For convenient selection oftransformants of the microorganism with the alteration in its genome thealteration may, e.g. be a DNA sequence encoding an antibiotic resistencemarker or a gene complementing a possible auxotrophy of themicroorganism.

Expression of a component of the respiratory chain may also be preventedor reduced by introduction of a DNA sequence complementary to the DNAsequence encoding the component at any genetic locus of themicroorganism, so as to prevent or reduce the expres-sion of thecomponent by an antisense mechanism.

As used herein, “target fermentation product” means a compound producedby fermenta-tion, such as for example riboflavin, pantothenic acid,biotin, thiamin, folic acid, pyrid-oxine, and amino acids.

As used herein, “cultivation of an aerobic microorganism to produce atarget fermentation product” means that the microorganism is inoculatedinto a fermentation medium supplied with all the substrates required forgrowth of the organism and production of the fermentation product. Theinoculated fermentation medium is subjected to certain physico-chemicalparameters, such as temperature, pH and aeration, that will allowopti-mal biomass growth and product accumulation. These parameters varyfrom type to type of microorganism to be cultivated and from compound tocompound to be produced. Procedures to empirically determine theseparameters are well-known to those skilled in the art and includefactorial plan or composite design. To further increase fermentationproduct accumulation substrates required for biomass growth or productformation may be supplied to the fermentation broth during the course ofthe cultivation of the micro-organism. For example, in the processaccording to the invention the microorganism may be subjected tofed-batch cultures with exponential and constant feeding profiles andchemostat cultures.

The fermentation process may be followed by analytically determiningprocess parameters. For example, cell dry weight (cdw) may bedetermined, e.g., from cell suspensions that are harvested bycentrifugation, washed with distilled water, and dried at, e.g. 110° C.for 24 h to a constant weight. Concentrations of carbon dioxide andoxygen in the bioreactor feed and effluent gas may be determined with,e.g., a mass spectrometer (e.g. Prima 600, Fisons Instruments). Glucoseconcentrations may be determined, e.g., enzymatically with, e.g.,commercial kits (e.g. Beckman).

Concentrations of organic acids, acetoin, and diacyl in the culturesupernatant may be determined by, e.g., HPLC on a Supelcogel C610Hcolumn (4.6×250 mm) (Sigma) with, e.g., a diode array detector (PerkinElmer). 0.2 N phosphoric acid may be used as mobile phase at a flow rateof 0.3 ml min-1 and 40° C. Target fermen-tation product concentrationsmay be determined by standard methods, e.g. riboflavin concentrationsmay be determined as, e.g., the absorption at 440 nm (A440) in cell-freeculture broth. If A440 exceeds 0.6, the broth may be diluted with, e.g.,0.5 M potassium phosphate buffer (pH 6.8). If A440 exceeds 1.8, forexample 0.8 ml of broth may be mixed with 0.2 ml of 0.2 M NaOH anddiluted to an appropriate concentration with 0.5 M potas-sium phosphatebuffer (pH 6.8).

The target fermentation product may be isolated from the microorganismand/or the me-dium. As used herein, the term “isolated” means that thetarget fermentation product is purified, or at least partially purifiedby methods including for example, filtration, centrifu-gation, and/orextraction. The target fermentation product may be further purified byre-crystallization from aqueous or organic solvents or applying othermethods known in the art, such as for example, ion-exchange,size-exclusion, or hydrophobic interaction chroma-tography. For adetailed description of the procedures for isolation and purificationof, e.g. riboflavin from a fermentation broth, see, e.g., EP 730,034.

The present invention further provides a polynucleotide, whichpolynucleotide is capable of preventing or reducing the expression of aless energy efficient component of the respi-ratory chain in a Grampositive host strain with alternative subchains of the respiratorychain. Preferably the Gram positive host strain is B. subtilis or C.glutamicum and the com-ponent of the respiratory chain, the expressionof which is to be prevented or reduced, is the terminal oxidase encodedby cydAB. Preferably the polynucleotide has a nucleotide sequence whichis illustrated as SEQ ID NO:1.

SEQ ID NO:1 may be modified at its 3′ and 5′ ends with extensionsequences, each of which are several hundred base pairs in length, toincrease the transformation efficiency of SEQ ID NO:1. The extensionsequences are random sequences, which are preferably less than 80%homologous to DNA sequences of the recipient cells to preventrecombination at undesired loci. Such a polynucleotide sequence is thenused to transform a micro-organism capable of producing a targetfermentation product.

The polynucleotide sequence of the present invention may, e.g. comprisea DNA fragment from the cyd locus of B. subtilis or C. glutamicumprovided with deletion-insertion muta-tions, e.g. as set forth in moredetail in the examples. The two subunits of the cytochrome bd oxidaseare encoded by cydA and cydB. In B. subtilis cydA and cydB comprise anoperon together with cydC and cydD at 340o of the B. subtilis genome.

Another embodiment of the present invention is a Bacillus subtilis hostcell or a Corynebac-terium glutamicum host cell transformed with apolynucleotide, which polynucleotide is capable of preventing orreducing the expression of the cytochrome bd terminal oxidase of therespiratory chain in the host cell.

For example, a polynucleotide sequence encoding a cyd operon of B.subtilis with an in-serted antibiotic resistance gene that replaces 1376bp from the 3′ end of cydB and the 5′ end of cydC may first beconstructed in E. coli. Transformation of a natural competent B.subtilis microorganism with the polynucleotide sequence results in a B.subtilis mutant pro-vided with a cyd deletion. A PBS1 phage lysateprepared from this mutant may then be used to introduce via generalizedtransduction the cyd deletion into the production micro-organism RB50containing multiple copies of the engineered rib operon pRF69. Standardrecombinant DNA techniques may be used for the construction of thepolynucleotide sequence and the B. subtilis mutants.

Transformants positive for the deletion-insertion mutation are selectedusing standard selection protocols. For example, the polynucleotidesequence used to transform the microorganism may include variousselection markers, including for example antibiotic resistance markers,color producing markers, etc. Preferably, the marker is a neomycinresistance marker, and selection for the desired transformation includesidentifying microorganisms capable of growing in fermentation mediasupplemented with neomycin, and which over-produce the targetfermentation product, such as riboflavin.

Preferably the aerobic microorganism with increased efficiency of therespiratory chain is a recombinantly produced microorganism thatover-produces riboflavin. As used herein, the term “over-produce” meansthat the microorganism produces the target fermentation pro-duct from asubstrate that is used as a carbon source above at least 0.1% (w/w)yield, pre-ferably above 1% (w/w) yield, such as for example, above 4%(w/w) yield.

An example of such aerobic host cell is a riboflavin producing B.subtilis RB50 cell, designated as RB50::[pRF69]n containing multiple (n)copies (e.g., about 5 to about 20 copies) of pRF69 encoding a rib operonmodified with the strong phage SPO1 promoter (P15) to enhancetranscription of the rib genes. This recombinantly-producedmicro-organism produces significantly more riboflavin than wild-typemicroorganisms.

B. subtilis RB50 was deposited with the Agricultural Research CultureCollection (NRRL), Peoria, Ill., under the terms of the Budapest Treatyon May 23, 1989, and was assigned accession number B 18502. PlasmidpRF69 was deposited with the American Type Culture Collection (ATCC),Rockville, Md., on Jun. 6, 1990, and was assigned accession number ATCC68338.

The present invention also includes derivatives of RB50::[pRF69]. Asused herein, a “deri-vative” of RB50::[pRF69] is any B. subtilis cellwhich contains the engineered rib operon of pRF69 or a polynucleotidesequence that is at least 25% identical to the engineered rib operon ofpRF69, preferably at least 50% identical to the engineered rib operon ofpRF69, and any other genetic modification, that leads to alterations inthe expression of the ribo-flavin biosynthetic genes. In the presentinvention, the percent identity of the polynucleo-tide sequence aredetermined using the BLAST program and the server at the National Centerof Biotechnology Information (Bethesda, Md., USA). A “derivative” ofRB50::[pRF69] may also contain alterations in the genome ofRB50::[pRF69], that affect the biosynthesis of compounds that arerequired as precursor compounds for riboflavin biosynthesis.Furthermore, auxotrophic RB50::[pRF69] mutants are also considered“derivatives” of RB50::[pRF69]. The term auxotrophic mutant refers to amicroorganism that has been modified, by e.g. a mutation, to require theaddition of an exogenous com-pound to grow, that prior to the mutationthe microorganism could produce itself.

Accordingly, the present invention provides

-   -   (1) a process for the preparation of a target fermentation        product comprising cultivation of an aerobic microorganism to        produce such product, wherein in the microorganism the        efficiency of the respiratory chain is increased, preferably by        introduction of a more energy efficient component or prevention        or reduction of a less energy efficient component of a subchain,        and the aerobic microorganism is a member of the bacterial        genera Escherichia, Bacillus, Cyanobacter, Streptomyces, and        Corynebacterium, preferably E. coli, B. subtilis, B.        amyloliquefaciens, B. licheniformis, B. ammoniagenes or C.        glutamicum;    -   (2) a process for the preparation of a target fermentation        product, e.g. riboflavin, panto-thenic acid, biotin, thiamin,        folic acid, pyridoxine, or an amino acid, comprising        cultiva-tion of an aerobic microorganism to produce such        product, wherein in the microorganism the efficiency of the        respiratory chain is increased;    -   (3) a polynucleotide, which polynucleotide is capable of        preventing or reducing the expres-sion of a less energy        efficient component of the respiratory chain, e.g. the terminal        oxidase encoded by cydAB, in a Gram positive host strain,        e.g. B. subtilis or C. glutamicum, with alternative subchains of        the respiratory chain;    -   (4) a nucleotide sequence which is illustrated as SEQ ID NO:1        which is optionally modified at its 3′- and 5′ ends with        extension sequences, each of which are several hundred base        pairs in length, and which extension sequences are random        sequences, less than 80% homologous to DNA sequences of the        recipient cells;    -   (5) a polynucleotide comprising a DNA fragment from the cyd        locus of B. subtilis or C. glutamicum provided with        deletion-insertion mutations;    -   (6) a Bacillus subtilis host cell or a Corynebacterium        glutamicum host cell transformed with a polynucleotide, which        polynucleotide is capable of preventing or reducing the        expression of the cytochrome bd terminal oxidase of the        respiratory chain in the host cell, e.g. wherein the        polynucleotide sequence encodes a cyd operon of B. subtilis with        an inserted antibiotic resistance gene that replaces 1376 bp        from the 3′ end of cydB and the 5′ end of cydC, and optionally a        further selection marker like an antibiotic resistance marker,        e.g. neomycin resistance marker, or a color producing marker;    -   (7) a host cell as in (6) which is a recombinantly produced        microorganism that over-pro-duces riboflavin;    -   (8) a host cell as in (6) wherein the microorganism produces the        target fermentation pro-duct from a substrate that is used as a        carbon source above at least 0.1% (w/w) yield, pre-ferably above        1% (w/w) yield, such as for example, above 4% (w/w) yield;    -   (9) a host cell as in (6) which is a B. subtilis RB50 cell,        designated as RB50::[pRF69]n con-taining multiple (n) copies        (for example about 5 to about 20 copies) of pRF69 encoding a rib        operon modified with the strong phage SPO1 promoter (P15) to        enhance transcription of the rib genes; and    -   (10) a host cell as in (6) which is a derivative of        RB50::[pRF69], e.g. a B. subtilis cell which contains the        engineered rib operon of pRF69 or a polynucleotide sequence that        is at least 25% identical to the engineered rib operon of pRF69,        preferably at least 50% identical to the engineered rib operon        of pRF69.

The following examples are set forth to illustrate the processes,polynucleotides and host cells of the present invention. These examplesare illustrative only and are not intended to limit the scope of theinvention in any way. For example, the present invention may be variedby carrying out a fermentation process to produce a target fermentationproduct with any microorganism having a natural capability to utilizealternative respiratory chains wherein expression of a component of arespiratory chain is prevented. According to the present invention theprocess can be carried out as a continuous culture or as a batch or fedbatch process in large scale industrial fermentors, varying the dilutionrate from 0.3 l/h to 0.001 l/h, increasing the concentration of thecomponents in the fermentation medium, or increasing glucoseconcentration up to 400 g/l. The media components and the re-quiredphysico-chemical parameters for all of these variations, would bedetermined and adjusted by one skilled in the art.

EXAMPLE 1 Construction of a Respiratory Mutant of B. subtilisRB50::pRF69 Carrying a cyd Deletion

Construction of a cydBC deletion-insertion mutation: A 3.4 kb DNAfragment is amplified from DNA of B. subtilis microorganism 1012 [Saitoet al., Mol. Gen. Genet. 170:117-122 (1979)] using primers CydA+1illustrated as SEQ ID NO:2 and CydC−1 illustrated as SEQ ID NO:3 and PCRreaction conditions of 30 cycles of denaturation at 95° C. for 1 min.,annealing at 50° C. for 1 min. and extension at 72° C. for 4.5 min. ThePCR product is puri-fied using the Wizard PCR purification kit (PromegaCorp.). The PCR product is ligated into the pGEM-TEasy vector (PromegaCorp.), resulting in plasmid pNMR20.

The 1.2 kb neomycin-resistance cassette from plasmid pBEST501 [Itaya etal., Nucl. Acids. Res. 17:4410 (1989)] is amplified using primerspBESTBcl+1 illustrated as SEQ ID NO:4 and pBESTBcl−1 illustrated as SEQID NO:5 using PCR reaction conditions as above. The amplifiedneomycin-resistant cassette is purified and digested with BclI, and iscloned into BclI-digested pNMR20 to give plasmid pNMR21 illustrated asSEQ ID NO:1 which con-tains the neo cassette inserted into cydBC in thesame orientation as cyd transcription. Plasmid pNMR21 is linearised withPstI and transformed into B. subtilis wild-type strain 1012 and selectedon TBAB plates containing neomycin to a final concentration of 5 mg×ml-1to give B. subtilis microorganism NM 18.

B. subtilis microorganism NM18 is used as a donor microorganism forpreparation of PBS1 phage lysate. This lysate is used to transduce theriboflavin production microorganism RB50 provided with the modifiedriboflavin operon pRF69. RB50 refers to the host micro-organism of B.subtilis, which contains several mutations introduced to improveproduc-tion of nucleotides and riboflavin. pRF69 refers to a rib operonmodified by the introduc-tion of strong phage promoters which isintroduced at the rib locus of pRF50. The modi-fied operon pRF69 isamplified to high copy numbers. A detailed description of themicro-organism RB50 and the modified rib operon pRF69 is presented EP405,370. A number of neomycin-resistant colonies are obtained. Three ofthese clones are analyzed by PCR and Southern hybridization, and areshown to contain the cyd deletion. One of these clones is selected andrenamed RB50::[pRF69] DcydBC. Southern blot hybridization reveals thepre-sence of pRF69.

RB50::[pRF69] DcydBC is cultivated in a rich, complex medium (VY medium:25 g/l of Difco veal infusion plus 5 g/l yeast extract) supplementedwith 10 mg/ml chloramphenicol to an optical density OD 660=1. Onemilliliter of this broth is transferred into 20 ml of VY mediumsupplemented with 30 mg/ml chloramphenicol and after reaching OD 1,again 1 ml of culture is transferred into 20 ml VY medium supplementedwith 60 mg/ml chlor-amphenicol. The same passage is repeated using VYcontaining 80 mg/ml chlorampheni-col. After reaching an OD of 1, thisculture is supplemented with 15% (Vol/Vol) glycerol and 1 ml aliquotsare frozen at −80° C. The stepwise increase in the antibioticconcentra-tion is used to select for bacteria with increased copy numberof the modified rib operon pRF69 (EP 405,370).

EXAMPLE 2 Fed-Batch Cultivation of RB50::[pRF69] DcydBC and the ParentStrain RB50::[pRF69]

For preparation of seed cultures aliquotes of the frozen RB50::[pRF69]DcydBC bacterial suspension of example 1 or the parent strainRB50::[pRF69] suspension are thawed and transferred into 100 ml VYmedium supplemented with 80 mg/ml chloramphenicol. The cultures areincubated at 37° C. until reaching OD=10 (typically after 12 to 15hours).

The main fermentation is initiated by inocculation of 50 ml of each ofthe seed cultures into 800 ml of a fermentation medium with thefollowing composition (per liter of ddH2O): 27.3 g glucose×H2O; 0.75 gNa glutamate; 0.23 g NH4Cl; 1.41 g (NH4)2SO4; 4.11 g Na2HPO4×2H2O; 4.71g KH2PO4; 4.71 g K2HPO4; 11.77 g yeast extract; 1 g MgSO4×7H2O; 62.5 mgCaCl2×2H2O; 40 mg FeSO4×7H2O; 14.6 mg MnSO4×H2O; 4 mg ZnSO4×7H2O; 0.8 mgCuCl2×2H2O; 4 mg CoCl2×6H2O; 0.3 mg Na2MoO4×2H2O; and 1 mg AlCl3×6H2O. Afeed pump is switched on shortly before glucose is depleted indicated bya the drop in CO2 production. The feed medium (655.2 g glucose×H2O; 1.5g MgSO4×7H2O; 11 mg MnSO4×H2O; and 3 mg ZnSO4×7H2O) is supplied at 13.3mL L-1 h-1 for 2 hours after initiation of the feeding phase and then at14.7 mL L-1 h-1. The fermentations are carried out in a 2 L LH discovery210 series reactor (Adaptive Biosystems) at 39° C. The stirrer speed isset to 1,500 rpm and the air flow is kept between 3 and 5 L min-1,ensuring dissolved oxygen level above 15% throughout the cultivation.

EXAMPLE 3 Riboflavin Production with RB50::[pRF69] DcydBC and the ParentStrain RB50::[pRF69]

200 μl of a 0.2N NaOH solution is added to 0.8 ml of the fermentationsamples of example 2 immediately after collection from the fermentationreactor. The sample is incubated for 20 seconds at room temperature todissolve riboflavin crystals within the sample. An ali-quot of thissuspension is diluted and neutralized with 0.5 molar potassium phosphatebuffer pH 6.8. The samples are centrifuged in a table top Eppendorffcentrifuge for 5 min at 14'000 rpm and the absorption at 440 nm (A440)in the supernatant is determined. The dilution of the samples isadjusted to achieve readings between 0.1 and 0.6 absorption units. Theriboflavin concentration is calculated by comparing the absorption ofthe samples to those of riboflavin standards (Sigma, St. Luis, Mo.,USA).

The results of this example show that upon introduction of the cyddeletion preventing the microorganism from using the cytochrome bdterminal oxidase subchain of the respiratory chain RB50::[pRF69] DcydBCproduces 15.5 g/l riboflavin compared to 14.0 g/l of the parent strainRB50::[pRF69] after 48 h of fermentation. At 24 hours of fermentationthe advantage of the cyd deletion strain over the parent strain is evenmore pronounced with 9.8 g/1 and 7.5 g/1, respectively.

1. A process for the preparation of a target fermentation productcomprising cultivation of an aerobic microorganism to produce suchproduct, wherein in the microorganism the efficiency of the respiratorychain is increased by introducing a polynucleotide into saidmicroorganism, said polynucleotide being capable of preventing orreducing the expression of a less energy efficient component of therespiratory chain in said microorganism.
 2. A process according to claim1 wherein the aerobic microorganism is a member of the bacterial generaEscherichia, Bacillus, Cyanobacter, Streptomyces, and Corynebacterium.3. A process according to claim 1 wherein the target fermentationproduct is riboflavin, pantothenic acid, biotin, thiamin, folic acid,pyridoxine, or an amino acid.
 4. A polynucleotide, which polynucleotidehaving the nucleotide sequence of SEQ ID NO: 1 and which is capable ofpreventing or reducing the expression of a less energy efficientcomponent of the respiratory chain in a Gram positive host strain withalternative subchains of the respiratory chain.
 5. A polynucleotideaccording to claim 4 wherein the Gram positive host strain is B.subtilis or C. glutamicum.
 6. A polynucleotide according to claim 4wherein SEQ ID NO:1 is modified at its 3′- and 5′ ends with extensionsequences, each of which are several hundred base pairs in length.
 7. ABacillus subtilis host cell or a Corynebacterium glutamicum host celltransformed with a polynucleotide, which polynucleotide is capable ofpreventing or reducing the expression of the cytochrome bd terminaloxidase of the respiratory chain in the host cell, wherein thepolynucleotide sequence encodes a cyd operon of B. subtilis with aninserted antibiotic resistance gene that replaces 1376 bp from the 3′end of cydB and the 5′ end of cydC.
 8. A host cell according to claim 7which is a recombinantly produced microorganism that over-producesriboflavin.